Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications

Jiahui Wu, Lin Liu, Tomoki Matsuda, Yongxin Zhao, Aleksander Rebane, Mikhail Drobizhev, Yu Fen Chang, Satoko Araki, Yoshiyuki Arai, Kelsey March, Thomas E. Hughes, Ken Sagou, Takaki Miyata, Takeharu Nagai, Wen Hong Li, Robert E. Campbell

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Original languageEnglish (US)
Pages (from-to)963-972
Number of pages10
JournalACS Chemical Neuroscience
Volume4
Issue number6
DOIs
StatePublished - Jun 19 2013

Fingerprint

Optogenetics
Artifacts
Chemical activation
Protein Engineering
Neocortex
Ions
Calcium
Cell culture
Light
Cell Line
Cells
Imaging techniques
Proteins

Keywords

  • Ca imaging
  • Ca indicators
  • channelrhodopsin
  • fluorescent proteins
  • photoactivation

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Physiology
  • Cognitive Neuroscience

Cite this

Wu, J., Liu, L., Matsuda, T., Zhao, Y., Rebane, A., Drobizhev, M., ... Campbell, R. E. (2013). Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications. ACS Chemical Neuroscience, 4(6), 963-972. https://doi.org/10.1021/cn400012b

Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications. / Wu, Jiahui; Liu, Lin; Matsuda, Tomoki; Zhao, Yongxin; Rebane, Aleksander; Drobizhev, Mikhail; Chang, Yu Fen; Araki, Satoko; Arai, Yoshiyuki; March, Kelsey; Hughes, Thomas E.; Sagou, Ken; Miyata, Takaki; Nagai, Takeharu; Li, Wen Hong; Campbell, Robert E.

In: ACS Chemical Neuroscience, Vol. 4, No. 6, 19.06.2013, p. 963-972.

Research output: Contribution to journalArticle

Wu, J, Liu, L, Matsuda, T, Zhao, Y, Rebane, A, Drobizhev, M, Chang, YF, Araki, S, Arai, Y, March, K, Hughes, TE, Sagou, K, Miyata, T, Nagai, T, Li, WH & Campbell, RE 2013, 'Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications', ACS Chemical Neuroscience, vol. 4, no. 6, pp. 963-972. https://doi.org/10.1021/cn400012b
Wu, Jiahui ; Liu, Lin ; Matsuda, Tomoki ; Zhao, Yongxin ; Rebane, Aleksander ; Drobizhev, Mikhail ; Chang, Yu Fen ; Araki, Satoko ; Arai, Yoshiyuki ; March, Kelsey ; Hughes, Thomas E. ; Sagou, Ken ; Miyata, Takaki ; Nagai, Takeharu ; Li, Wen Hong ; Campbell, Robert E. / Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications. In: ACS Chemical Neuroscience. 2013 ; Vol. 4, No. 6. pp. 963-972.
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